Biofilms, organized aggregates of matrix-associated bacteria, enhance the ability of bacteria to colonize surfaces, including host tissues. Bacteria in biofilms are responsible for the majority of hospital-acquired infections, including those stemming from medical devices such as catheters, and exhibit substantially increased resistance to anti-microbials, thus diminishing the effectiveness of antibiotic treatment. While numerous bacteria are currently being studied for their ability to form and disperse from biofilms, we are able to examine the role of biofilm formation by Vibrio fischeri both in laboratory culture and in a natural animal model of infection. We have shown that biofilm formation represents a critical early step during initiation by V. fischeri of symbiotic colonization of its host, the squid Euprymna scolopes. Both biofilm-like aggregation on the surface of the symbiotic organ and subsequent colonization depend upon an 18 gene cluster (syp) that we have recently discovered as well as its regulators. The syp cluster includes polysaccharide biosynthesis genes and several novel regulators. Induction of syp enhances symbiotic biofilm formation and colonization, while loss of syp disrupts both. Strikingly, these in situ colonization phenotypes are tightly correlated with biofilm phenotypes readily observable in laboratory culture. This model thus affords us an exception opportunity to develop and test hypotheses about the role of biofilms in bacterial colonization of a eukaryotic host through genetic and biochemical analysis of this locus and its regulators. To date, we have uncovered a complex regulatory circuitry used by V. fischeri to control biofilm formation, including both activators and inhibitors, thus making it a rich model for understanding how biofilm formation can be controlled. Control of biofilm formation, as well as additional responses of the bacterium to its host, are key components in understanding the developmental changes that enable V. fischeri to successfully navigate natural barriers to colonization. We therefore propose to further explore how V. fischeri responds to its host, and particularly ask how induction of the syp locus, essential for biofilm formation, is regulated (Aim 1). In addition, we will ask how biofilm formation is controlled by the novel response regulator SypE, which (a) plays both positive and negative roles in biofilm formation, (b) is predicted to express serine kinase and phosphatase activities, and (c) is likely controlled through a signal transduction cascade (Aim 2). Finally, our evidence suggests that other factors contribute to biofilm formation, and thus we propose to identify these factors and determine their roles in symbiotic biofilm formation and colonization (Aim 3). In each of the aims, we propose to examine the correlation between phenotypes observed in vivo with those that occur in situ. This powerful tool, the ability to compare in vivo and in situ biofilm phenotypes, combined with the study of a model organism whose biofilm formation capability is under complex regulatory control, has the potential to reveal insights into environment-specific control not yet identified by the more traditional models of biofilm formation. PUBLIC HEALTH RELEVANCE: Bacterial cells can associate with themselves and other bacteria in microbial communities called biofilms, which exhibit increased resistance to anti-microbial therapies such as antibiotics. While biofilms are being intensively studied in the laboratory, few models exist in which biofilm formation in the lab can be compared to those that occur naturally in an animal host. One of these is our model, the symbiosis between Vibrio fischeri and its squid host, which has revealed a clear correlation between biofilms in lab and those formed during bacteria-host interactions, as well as complex regulatory control that we propose to investigate further here.